When an adaptive immune response occurs in an exaggerated or inappropriate form, the term allergy or hypersensitivity is applied. Allergic or hypersensitivity reactions are the result of normally beneficial immune responses acting inappropriately to foreign antigens (usually environmental macromolecules) and sometimes cause inflammatory reactions and tissue damage. In these situations, a normally harmless environmental stimulus, called an “allergen”, triggers an immune response which upon re-exposure, is re-activated to generate pathological damage. Allergies or hypersensitivities are distinguished into four types of reactions. The first three are antibody-mediated, the fourth is mediated mainly by T cells and macrophages.
In Type I Immediate Hypersensitivity/Atopic Allergy, the principal immune response to the allergen involves the production of IgE antibodies. Such disorders are by far the most prevalent in humans and are seen as principal targets for new therapeutic approaches. Although these diseases are not exclusively IgE mediated, IgE binds to cells within the tissues such as mast cells and basophils and the cross-linking of IgE on the cells surfaces by allergen invokes the release of many inflammatory mediators.
Typical examples of such diseases include asthma, allergic cough, allergic rhinitis and conjunctivitis, atopic eczema and dermatitis, urticaria, hives, insect bite allergy, dietary and certain drug allergies. In many cases, the particular allergens are known. By way of example, the principal allergen in asthma is DerP1 from house dust mite but other triggers of asthma such as pet dander antigens also exist.
Type II or antibody dependent cytotoxic hypersensitivity occurs when antibodies of a different type, usually IgG and IgM, binds to either self antigen or foreign antigen on cells and leads to phagocytosis, killer cell activity or complement mediated lysis. These types of allergies are relatively unusual but can include some allergies to drugs.
Type III hypersensitivity develops when immune complexes are formed in large quantities or cannot be cleared adequately by the reticuloendothelial system. The immune complexes usually result from the deposition of antibody, usually IgM or IgG, allergen complexes at these sites. In normal circumstances, antibody binds to allergen and is cleared by a variety of tissue cells. However, a number of factors may influence the persistence of the immune complexes and where they remain in the blood for prolonged periods, they can lodge and establish inflammation in the kidneys, skin (where they cause rashes) and joints (where they can cause a type of arthritis other than rheumatoid arthritis).
Type IV or delayed type hypersensitivity (DTH) does not involve antibody but instead the prolonged activation of T lymphocytes. These T cells are capable of secreting soluble factors causing tissue damage and enhancing the recruitment and activation of other cell types to the tissues. Incoming cells themselves contribute to the inflammation and tissue damage. DTH is most seriously manifested when antigens (for example those associated with mycobacteria tuberculosis) are trapped in a macrophage and cannot be cleared. T cells are then stimulated to elaborate cytokines which mediate a range of inflammatory responses. DTH reactions are less common than Type I reactions but are seen in graft rejection and allergic contact dermatitis which is generally manifested as a contact sensitivity (allergy usually involving skin rash) to environmental “contact allergens” such as heavy metals.
Oral administration of antigens—such as allergens and autoantigens—has long been recognised as a method to prevent peripheral T cell responses and, in the case of autoantigens, has also been shown to prevent or delay the onset of several experimental autoimmune diseases including experimental allergic encephalomyelitis (EAE). Major problems recognised with such strategies are that it usually requires feeding of large, if not massive, doses of autoantigens and it is generally less efficient in an immune as opposed to a naive host. The latter problem has limited the therapeutic potential of this strategy. However, it has now been shown by Sun et al (1994 Proc Natl Acad Sci 91: 10795-10799) that oral administration of minute amounts of prototype particulate and soluble protein antigens conjugated to cholera toxin B subunit (CtxB), the nontoxic receptor-binding moiety of cholera toxin, can readily induce tolerance in the peripheral T-cell compartment and is effective not only in naive but also in systemically sensitised animals. In addition, oral administration of minute amounts of an autoantigen, myelin basic protein (MBP), coupled to CtxB can prevent EAE in Lewis rats (Sun et al 1996 Proc Natl Acad Sci 93: 7196-7201). Other researchers have also shown that feeding even a single dose of minute amounts (microgram) of antigens conjugated to the receptor binding nontoxic B subunit moiety of cholera toxin (CtxB) can markedly suppress systemic T cell mediated inflammatory reactions in naive as well as in experimental animals (Bergerot et al 1997 Proc Natl Acad Sci 94: 4610-4614).
Escherichia coli (E. coli) heat labile enterotoxin (Etx) and its closely related homologue, cholera toxin (Ctx), are examples of protein toxins which bind to glycolipid receptors on host cell surfaces. Each toxin consists of six noncovalently linked polypeptide chains, including a single A subunit (27 kDa) and five identical B subunits (11.6 kDa) which principally bind to GM1 ganglioside receptors found on the surfaces of mammalian cells (Nashar et al 1996 Proc Natl Acad Sci 93: 226-230). The A subunit is responsible for toxicity possessing adenosine diphosphate (ADP) ADP-ribosyltransferase activity, whereas the B subunits (EtxB and CtxB) are non-toxic oligomers which bind and cross-link a ubiquitous cell surface glycolipid ganglioside, called GM1, thus facilitating A subunit entry into the cell.
The GM1 ganglioside receptor is a member of family of gangliosides comprising sialic acid containing glycolipids (also called glycosphingolipids) which are formed by a hydrophobic portion, the ceramide, and a hydrophilic part, that is the oligosaccharide chain. Gangliosides are defined as any ceramide oligosaccharide carrying, in addition to other sugar residues, one or more sialic residues (Oxford Dictionary of biochemistry and molecular biology. Oxford University Press. 1997. Eds Smith A D, Datta S P, Howard Smith G, Campbell P N, Bentley R and McKenzie H A). Although first described in neural tissue, several studies have shown that gangliosides are almost ubiquitous molecules expressed in all vertebrate tissues. Within cells, gangliosides are usually associated with plasma membranes, where they may act as receptors for a variety of molecules and take part in cell-to-cell interaction and in signal transduction. In addition, gangliosides are expressed in cytosol membranes like those of secretory granules of some endocrine cells such as the pancreatic islets and adrenal medulla.
Gangliosides contain in their oligosaccharide head groups one or more residues of a sialic acid which gives the polar head of the gangliosides a net negative charge at pH 7.0. The sialic acid usually found in human gangliosides is N-acetylneuraminic acid. Over 20 different types of gangliosides have been identified, differing in the number and relative positions of the hexose and sialic residues which form the basis of their classification. Nearly all of the known gangliosides have a glucose residue in glycosidic linkage with ceramide, residues of D-galactose and N-acetyl-D-galactosamine are also present.
In the ganglioside nomenclature of gangliosides, devised by Svennerholm (Biochemistry Lehninger 2nd Ed 1975 Worth Publishers Inc p 294-295), the subscript letters indicate the number of sialic groups. M is monosialo, D is disialo and T is trisialo.
One of the best studied members of the ganglioside family is the monosialosylganglioside, GM1, which has been shown to be the natural receptor for the cholera toxin. Soluble ganglioside GM1 binds to the toxin with high affinity and inactivates it (Svennerholn 1976 Adv Exp Med Biol 71: 191-204).
The chemical formula for GM1 can be represented as:Gal β3GalNAc β4(NeuAc alpha 3)Gal β4Glc β1 Cerwhere Glc is D-glucose, Gal is D-galactose, GalNAc is N-acetyl-D-galactosamine; NeuAc is N-acetylneuraminic acid, Cer is ceramide.
The chemical formula for GM1 can also be represented asgalactosyl-N-acetylgalactosaminyl {sialosyl} lactosyl ceramideorgalactosyl-N-acetyl-galactosaminyl-(sialyl)-galactosylglusosylceramide
The x-ray crystal structures of Etx bound to lactose (Sixma et al 1992 Nature (London) 355: 561-564) and CtxB bound to the pentasaccharide of GM1 (Merritt et al 1994 Protein Sci 3: 166-175) have revealed that CtxB and EtxB bind to the terminal galactose and sialic acid moieties of GM1 which can be represented asGalβ-1-3-3GalNAcand that such binding does not induce any striking changes in B subunit conformation.
Furthermore the cholera toxin has been shown to demonstrate an absolute requirement for terminal galactose and internal sialic acid residues (as in GM1) with tolerance for substitution with a second internal sialic acid (as in GD1b).
Etx, like Ctx also probably binds to the terminal sugar sequenceGal β1-3GalNAc β1-4(NeuAc alpha 2-3)Galwhere GalNAc is the N-acetylgalactosamine and NeuAc is N-acetylneuraminic acid.
In addition to binding to GM1, EtxB binds weakly to other gangliosides, including non-galactose containing GM2 and asialo-GM1 as well as galactoproteins (Nashar et al Immunology 1997 91: 572-578). Other researchers have shown that EtxB is capable of binding to GM1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-GM1 and GD1b respectively) but not substitution of the terminal galactose of GM1 (Umesaki and Setoyama 1992 Immunology 75: 386-388).
In contrast to the poor immunogenicity of the A subunit alone, both EtxB and CtxB are exceptionally potent immunogens and their respective holotoxins, Etx and Ctx, are known to be exceptionally potent adjuvants when given orally in combination with unrelated antigens (Ruedl et al 1996 Vaccine 14: 792-798; Nashar et al 1993 Vaccine 11: 235; Nashar and Hirst 1995 Vaccine 13: 803; Elson and Ealding 1984 J Immunol 133: 2892; Lycke and Holmgren 1986 Immunology 59: 301). Because of their remarkable immunogenicity, both EtxB and CtxB have been used as carriers for other epitopes and antigens (Nashar et al 1993 ibid) and have been used as components of vaccines against cholera and E. coli diarrhoea (Jetborn et al 1992 Vaccine 10: 130).
The ability of the B subunit of Ctx and Etx to interact with receptors present on mammalian cells has been shown to exert modulatory effects on the function of those cells. It is known that cells of the immune system are differentially affected following such interaction. In particular, WO 95/020045 discloses that EtxB binds to GM1 ganglioside receptors which are found on the surfaces of mammalian cells and that this binding induces differential effects on lymphocyte populations including a specific depletion of CD8+ T cells and an associated activation of B cells. These effects are absent when a mutant EtxB protein lacking GM1 binding activity is employed. These observations have led to the use of agents capable of binding to GM1 in the prevention and treatment of autoimmune disease, transplant rejection and graft versus host disease (GVHD). These studies suggest that agents that bind to GM1 or mimic binding to GM1 promote the induction of immunological tolerance.
Researchers have shown that a state of immunological unresponsiveness, also known as “immunological or oral tolerance”, can be induced by the oral administration of dietary protein antigens. (Sun et al 1994 ibid; Sun et al 1996 ibid, Bergerot et al 1997 ibid). The inhalation of antigens can also induce a state of specific immunological unresponsiveness or “nasal tolerance”. Thus, systemic immunological tolerance can be induced when antigen is administered orally or nasally by a mucosal route. WO 95/01301 discloses an immunological tolerance-inducing agent comprising a mucosa-binding agent linked to a specific tolerogen. WO95/10301 also includes mention of the treatment of allergy using a mucosa binding agent coupled to an allergen. Other researchers such as Tamura et al (1997 Vaccine 15: 225-229) have taken directly the protocol of WO 95/10301 and tested its efficacy in preventing allergy in a murine model of Type I allergy. They reported a significant lowering of IgE levels which are a strong predictor of efficacy and they cite data, following administration of EtxB coupled to ovalbumin (the results were not included), which shows that EtxB was not effective once IgE levels are established. It has also been shown that orally administered Ctx and Etx can act on several humoral and cellular immune responses not only at the gastrointestinal tract, but also in distant mucosal effector sites such as the respiratory tract. These data suggest that these mucosal adjuvants have a potential use in oral immunisation strategies to improve the local immune responses in remote mucosal tissues, in accordance with the concept of a common mucosal immune system (Bienenstock J 1974 The physiology of the local immune system and the gastrointestinal tract. In: Progress in Immunology II, vol 4: clinical aspects, I. L. Brent, J. Holborrow, Eds. Amsterdam, North Holland, pp197-207; Ruedl et al 1996 ibid; Umesaki 1992 ibid; Czerkinsky and Holmgren (1994 Cell Mol Biol 40: 3744).
The induction of immunological tolerance may include a number of different mechanisms which may be summarised as follows:                (i) a process whereby antigen reactive cells are removed through triggering them to commit suicide (apoptosis);        (ii) an induction of anergy or the long term inactivation of the antigen reactive cells;        (iii) immune deviation of the antigen reactive cells away from the production of pathological responses;        (iv) suppression of the antigen reactive cells or their regulation by specific factors or regulatory cells        
In the treatment of allergy, it is possible that the induction of any of these mechanisms may be useful. However, while the deletion of antigen reactive cells and/or the induction of anergy are useful strategies once the precise allergens are known, invoking these mechanims will usually silence only those cells which respond to the allergen which was given in the treatment regime. On the other hand, the implementation of immune deviation or suppression strategies has the advantage of potential regulation of responses to antigens which are involved in the condition but were not part of the treatment. This phenomenon, known as “bystander suppression” allows the “spread” of tolerance to other antigens (such as allergens) in the target tissues through either the possible secretion of non-antigen specific suppressor molecules or through suppressive cellular interactions in that tissue as a result of the interaction between the antigen specific cells and the specific immunising antigen. In this way, as long as at least one of the antigens involved in the disorder is known, the condition may be treated even if there are other antigens implicated as well. Thus, the goal of a good treatment is the induction of a specific immune deviation or suppression.
Nashar and co-workers (Proc Natl Acad Sci 1996 93: 223-226; Int Immunol 1996 8: 731-736; Immunol 1997 91: 572-578) have demonstrated that the administration of EtxB and other homologues can modulate the immune response away from the production of Th1 cytokines such as IFNγ and interleukin 2 (IL-2) and towards the secretion of Th2 cytokines such as IL-4, IL-10 and IL-13. IFNγ is the classical Th1 cytokine, IL-4 is the classical Th2 cytokine. This “immune deviation” is the basis of the disclosure in WO 97/02045 and has been shown to be effective in the treatment of autoimmune diseases. The experimental results in WO 97/02045 would suggest that GM1 binding agents would not find use in the treatment of allergic conditions and/or hypersensitivity conditions since such conditions involve IgE, the production of which is generally accepted to be promoted by IL-4 and down regulated by IFNγ.
The present invention now seeks to provide new ways of treating allergic conditions and/or hypersensitivity conditions through the induction of a specific immune deviation or suppression.